Gene knock out technology has been shown to be enormously valuable in biological research. Conventional gene disruption technology by homologous recombination is a laborious and unpredictable process. The targeted genome editing developed in recent years is much more effective and may be achieved by targeted double strand breakage (DSB) in chromosomes. Examples of this technique utilize Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR), zinc-finger nucleases (ZFN) or transcription activator-like effector nucleases (TALEN) (Esvelt and Wang, Mol. Syst. Biol., 2013, 9:641). Among these, CRISPR is the most versatile genome editing tool because of its targeting mechanism by specific RNA sequence complementary to the genome modification site. CRISPR permits genome editing in multiple sites by using a cluster of targeting RNA sequences. DSB in cells is repaired by non-homologous end joining (NHEJ), which in low frequency, results in small insertion or deletion (indel) that may create a frameshift and inactivate the gene of interest.
Using most methods, the rate of genome editing is relatively low, typically less than 5%. The most common methods of assessing genome editing, detecting duplex DNA mismatch utilizing the Surveyor® nuclease assay (Transgenomic, Inc., Omaha, Nebr.) or deep sequencing, do not differentiate single allele or double allele mutations. Since the possibility of producing a double allele mutation in a single step is negligible, 2-step sequential genome editing is usually required.
Therapeutic antibodies are a successful new class of drugs developed in the past two decades. Over thirty recombinant therapeutic antibodies have been approved by the FDA for the treatment of various diseases including cancer, viral infection, organ graft rejection, rheumatoid arthritis and other autoimmune conditions. Many more therapeutic antibodies are in clinical trials for an ever-widening variety of diseases. With the advent of molecular biology, it has become possible to produce recombinant antibodies in mammalian cells (N. Yamane-Ohnuki and M. Satoh, mAbs, 2009, 1(3):230-236).
Antibody therapy directed against soluble factors, such as vascular endothelial growth factor or tumor necrosis factor, aims simply to reduce the free ligand concentration by immunocomplex formation. In contrast, when antibody therapy is directed at cell surface antigens, as is usually the case in antineoplastic immunotherapy, the goal is typically the removal of the disease-causing cell itself. Antibody-dependent cellular cytotoxicity (ADCC), a lytic attack on antibody-targeted cells, is triggered upon binding of lymphocyte receptors (e.g., FcγRs) by the constant region (Fc) of the antibodies, in most cases, immunoglobulin subclass 1 (IgG1). ADCC is considered to be a major function of some of the therapeutic antibodies, although antibodies have multiple therapeutic functions (e.g., antigen binding, induction of apoptosis, and complement-dependent cellular cytotoxicity) (T. Shinkawa, et al., J. Bio. Chem., 2003, 278(5):3466-3473). In ADCC, natural killer (NK) cells recognize the constant (Fc) region of antibodies primarily via interaction with the NK cell's FcγRIII receptor, which then activates cell lysis and apoptosis.
The Fc-FcγRIII interaction is extremely sensitive to Fc glycosylation. Aglycosylated antibodies, e.g., those produced by non-mammalian cell lines, fail to bind Fc receptors (Leatherbarrow et al., Mol. Immun., 1985, 22:407-15; Walker et al., Biochem. J., 1989, 259:347-53; Leader et al., Immunology, 1991, 72:481-5). On the other hand, fucosylation of the carbohydrate chain attached to Asn297 of the Fc region reduces binding to FcγRIII and reduces in vitro ADCC activity (Shields et al., J. Biol. Chem., 2002, 277:26733-40; Shinkawa et al., J. Biol. Chem., 2003, 278:3466-73; Niwa et al., Cancer Res., 2004, 64:2127-33).
The majority of mammalian immunoglobulins are fucosylated, including those produced by Chinese hamster ovary cells (CHO cells) (Jefferis et al., Biochem J., 1990, 268:529-37; Raju et al., Glycobiology, 2000, 10:477-86). Fucose is attached to the Fc core region via an a-1,6 linkage generated by the α-1,6 fucosyltransferase (FUT8) protein (Oriol et al., Glycobiology, 1999, 9:323-34; Costache et al., J. Biol. Chem., 1997, 272:29721-8). Disruption of the Fut8 gene in CHO cells can eliminate core fucosylation of antibodies and can increase ADCC ˜100 folds (Yamane-Ohnuki et al., Biotech. Bioengin., 2004, 87(5):614-622).
Genome editing has been used to reduce or eliminate FUT8 activity by knocking down or knocking out both Fut8 alleles. Imai-Nishiya et al. have disclosed the double knock-down of Fut8 and GMD, using a siRNA tandem expression vector for targeting these genes and introducing it into IgG1 antibody-producing CHO/DG44 32-05-12 cells (BMC Biotechnology, 2007, 7:84; doi:10.1186/1472-6750-7-84). To create double allele Fut8 knock-out CHO cells, Yamane-Ohnuki et al. disclosed a two-step sequential homologous recombination process (Biotech. Bioengin., 2004, 87(5):614-622) to overcome the low frequency of homologous recombination. Similarly, Collingwood disclosed a targeted ZFN method to knock out both Fut8 alleles in CHOK cells by continuous culturing in the presence of a lethal dosage of Lens culinaris Agglutinin (LCA) to enrich Fut8 null cells, taking advantage of cell toxicity induced by specific binding of LCA to fucose (WO 2009/009086; L. Malphettes et al., Biotech. Bioengin., 2010, 106(5):774-783).
Further, it may be desirable to produce cells lines in which other genes in addition to the Fut8 gene are partially or fully suppressed. Because the rate of genomic editing is low, typically less than 5%, it is reasonable to assume that the possibility of producing a double allele mutation by a single crRNA site is negligible. One may have to perform sequential genome editing in order to obtain double allele knock-outs. For example, Cong, et al. disclosed successful genome editing in two endogenous genes (EMX1 and PVALB) and two targeting sites in the same EMX1 gene (Science, 2013, 339:819-823). However, the efficiency of deletion was only 1.6% when 2 protospacers in the EMX1 gene were targeted, and no double allele knock-outs were reported.